The T cell receptor (TCR), the signals that it transduces, and the molecules with which it interacts have long been known to be central to the functioning of lymphocytes and the immune system. Defects in these molecules or signaling pathways manifest in many disease states, including cancer and autoimmunity. Recently it has become apparent that plasma membrane domains known as 'lipid rafts', which are enriched in key signaling proteins and complexes, are also significantly involved in TCR-related signaling pathways. It thus seems clear that determination of which proteins or protein complexes associate with or dissociate from the macromolecular structures such as the TCR and lipid rafts, and in response to which modes of cell stimulation, will shed considerable new light on how these complexes regulate their critical immune cell functions. These questions will be addressed by the application of advanced mass spectrometry (MS)-based quantitative proteomics. Such methods allow for the simultaneous identification of potentially hundreds of proteins present in any given protein complex isolated from T cell cultures, and which proteins in the complex change in abundance, and by how much in response to any given stimulation. Modes of stimulation used will include via the TCR itself, as well as via TCR co-receptors CD28, CD4 and CD8, either individually or in combination. A number of cloned T cell lines expressing or lacking key selected T cell signaling molecules relevant to these studies will allow more precise determination of the role these molecules, and the other proteins with which they form complexes, play in T cell signaling. Proteins targeted for such studies will be molecules known to be critical for T cell signaling, and will include the adapter molecule LAT, the tyrosine kinase Lck, and newly identified proteins from this study. Furthermore, since protein phosphorylation is also known to regulate many biological signaling mechanisms, including regulation of TCR signaling, another new quantitative MS-based methodology will similarly be applied for determination of which of the proteins found to be associated with the TCR and lipid rafts are inducibly phosphorylated, on which residue(s), and how their phosphorylation state changes in response to stimulation. Such studies are expected to provide a thorough overview of the many proteins involved in TCR and lipid raft signaling, how the localization and phosphorylation of individual and groups of proteins are differentially regulated by the various modes of stimulation, and will identify many proteins not previously known to be involved in such processes. These identifications will provide useful new targets for follow-up biochemical, molecular biological and MS-based studies to further characterize T cell signaling events, ultimately leading to the possible identification of new markers for various disease states or targets for therapeutic intervention, and a more profound understanding of the mechanisms controlling and effecting T cell activation.